Different Mutations in mucA gene of Pseudomonas aeruginosa mucoid strains in cystic fibrosis patients and their effect on algU gene

expression

Giovana Pulcrano, Dora vita Iula, Valeria Raia, Fabio Rossano, Maria Rosaria Catania

Verónica Arango Baena Alejandra Baldiris Ardila

Third Semester – Molecular biologyUniversidad Pontificia Bolivariana

2012

IntroductionPseudomonas

Bacterium type bacilli.

Gram negative aerobic and anaerobic.

Bacterium with mobility by flagellum.

Easy cultivate in vitro.

IntroductionAeruginosa

Humans opportunistic pathogen that affect immunocompromised and hospitalized patients.

Gram Negative Aerobic with motility single- pole.

The most important diseases caused by P. aeruginosa are lung infections in patients with predisposing local causes such as mechanical ventilation or cystic fibrosis.

IntroductionCystic fibrosis

It is a inherited disease that caused the accumulation of thick, sticky mucus in the lungs, gastrointestinal tract and other body areas.

It is one type of chronic lung disease most common in children and young adults, and is a potentially fatal disorder.

IntroductionGen

Is an ordered sequence of nucleotides in the DNA contain the information necessary for the synthesis of a proteins and RNAs .

Gene mucA

MucA can also act as a regulator of algU transcription forming a

complex with MucB and altering the signal transduction pathway that maintains the low

transcriptional activity of algU

Gene AlgU

algU gene expression was analyzed to determinate if mutations in mucA.

IntroductionThe P. aeruginosa is a gram-negative bacteria of

the genus of Pseudomonas, which mainly affects immunocompromised patients such as cystic fibrosis, the genome of this bacterium has different mutations in the mucA gene affecting the expression of the algU gene.

GENERAL OBJECTIVE.Analysis of the influence of MucA gene

mutations in mucoid strains of P. aeruginosa on algU expression levels designed to establish if different mutations in the mucA gene resulting in length function as a controller of alginate production and swimming ability.

Métodos y Muestra Población de estudio

Se realizo en 119 cepas de P. aeruginosa, a partir de muestras respiratorias de pacientes con fibrosis quísticas. 145 hombres, 155 mujeres con una media de edad de 16.21 años.

La cepa de referencia que se

utilizo fue P. Aeruginosa

ATCC 27853.

Métodos y Muestra Extracción de DNA

El ADN genómico fue evaluado cuantitativamente por espectrofotometría y electroforesis.

La electroforesis es una técnica para la separación de moléculas en un campo eléctrico.

Métodos y Muestra PCR

Para identificar las mutaciones en el gen mucA se utilizo la PCR para obtener unos productos de amplificación.

PCR en tiempo real o cuantitativa

Se utiliza para amplificar y cuantificar de forma absoluta el producto de la amplificación del ADN

Métodos y Muestra Extracción de ARN

Mediante la utilización de ARNasa se hizo una hidrólisis del ARN con el fin de que este no interviniera en los resultados.

La actividad de la DNasa fue bloqueada por la adicción de calor, para desnaturalizarla y posteriormente analizar el resulta espectrofotométricamente.

Métodos y Muestra Movilidad

Este análisis se utilizo para buscar un indicativo de la presencia y función del flagelo.

Si se desplazaba mas de 6 mm era un indicativo positivo.

Métodos y Muestra PRGE ( electroforesis en gel de campos pulsantes)

se utiliza para examinar DNA cromosómico de organismos eucariotas.

en el estudio se utilizo para comparar las cepas y fueron consideradas diferentes si sus patrones de restricción difieren por 7 o mas bandas.

RESULTADOS

Discussions Authors

Publications Agreement or Disagreement

Wood & Ohman

A key elements of alginate operon regulation is the alternative sigma factor AlgU that interacts with MucA and acts as negative regulation of alginate biosynthesis: in fact, the occurrence of mutations in mucA was found to be the most frequent mechanism of conversion to the mucoid form.

Agreement

Boucher et al.

The results of real- time PCR showed that the expression of AlgU gene varied significantly in the three groups. In group II, algU gene expression level were similar to those of reference strains. It can be assumed that in this group, in which algU expression levels are low but there is also production of alginate, gene regulation occurs at different levels.

Agreement

Authors

Publications Agreement or Disagreement

Tart et al

algU was reported to affect not only the amount of alginate production, but also the expression of genes that control flagella functionality. The fleQ gene, involved in the regulation of the synthesis of the flagellum, was inhibited by the expression of the algU gene, thus in each of the strains analyzed a loss mobility inversely proportional to alginate production was expected.

Agreement

Tart et al

The complete inactivation of the flagellum with consequent loss of motility, however, was only observed in the two strains which had mucA novel mutations. In addition, PA91 non- mucoid strain grouped with group II instead of group I in the motility analysis.

Agreement

Discussions

Conclusions The Pathological action is closely related to the

characteristics of the strain P. aeruginosa: flagella, proteins and mucoid phenotype whether or not mucoid.

Conclusions To analyze the sequence of the mucA gene in all

strains was found that 40% (30) were mutated, which tells us that we find great variability of strains in one patient, although the percentage is not too high.

Conclusions

P.aeruginosa to be primarily responsible for respiratory illnesses, so this study on different strains opens the stakes to new clinical treatments.

Conclusions the study showed that alterations in the mucA

gene can alter the expression of the algU gene and thus affect the characteristics of the P.aeruginosa.

Alejandra Baldiris Ardila

Verónica Arango Baena